Many working environments contain viable and non-viable airborne microorganisms which may cause adverse health effects. Current assay methods rely on culturing techniques which only detect viable microorganisms and so result in underestimates of total concentrations of airborne microorganisms. This study investigated the use of a single antibody sandwich enzyme-linked immunosorbent assay (ELISA) for rapid, specific detection and enumeration of airborne microorganisms. ELISAs were developed for three microorganisms commonly used in the biotechnology industry (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae). These microorganisms were aerosolized and collected with an IOM personal sampler and a glass cyclone sampler. The resultant samples were assayed using the ELISAs. Results were compared to traditional culturing methods and light microscopy. The results indicated that ELISA provides a reliable, relatively rapid, specific and quantitative method for the detection of airborne microorganisms independent of their metabolic state.